Fig. 5

Activation of canonical Wnt signaling rescues MRF expression. a, b Scr- or shJarid2-expressing cells were differentiated for 2 days (D2) and shJarid2 cells were treated with either 10 mM NaCl or 10 mM LiCl while differentiating. Myod1 (a) and Myog (b) mRNA expressions were analyzed by qRT-PCR. Fold change in expression of mRNA was calculated relative to 10-mM NaCl-treated scr control (D2). Data are plotted as means with S.E.M. and analyzed with one-way ANOVA followed by Tukey’s HSD post hoc test. Means not sharing same letter are statistically significant (p < 0.001). n ≥ 4. c Cells as in a were analyzed for protein expression of MYOD and MYOG by western blot assays. Data are plotted as bar graphs with respect to the normalizer (GAPDH) in the right panel. Error bars are S.E.M. n ≥ 3. d, e Scr- or shJarid2-expressing cells were transiently transfected with an expression construct for Wnt3a (cDNA) or vector control and assayed for mRNA expression of Myog (d) and differentiation-specific genes (e) by qRT-PCR. UD represents undifferentiated myoblasts, and D2 represents 2 days of differentiation. Fold change was calculated relative to scr UD. Error bars are S.E.M. n ≥ 3. f JARID2 depletion impairs Wnt-mediated transcriptional activity and can be rescued by LiCl or WNT3A. Reporter assays were performed using TOPFlash and FOPFlash luciferase reporters in scr or shJarid2 cells. A set of shJarid2 cells was also transfected with an expression construct for Wnt3a (cDNA). Growth medium was replaced with differentiation medium with or without LiCl or NaCl 24 h post-transfection, and luciferase activity was detected 48 h post-transfection. Error bars are S.E.M. n ≥ 3